Nicotinamide adenine dinucleotides are involved in a very large number of oxidoreduction reactions both in the cytosol and in mitochondria. In general, they are not tightly bound to enzymes and are best considered as substrates, although they are often referred to as coenzymes. Nicotinamide adenine dinucleotide (NAD.sup.+) and nicotinamide adenine dinucleotide phosphate (NADP.sup.+) undergo reversible reduction to NADH and NADPH, respectively, but fulfill different roles in the cell. The major role of NADH is to transfer electrons from metabolic intermediates in a large number of biosynthetic processes into the electron transfer chain. NADPH acts as a reducing agent in a large number of biosynthetic processes.
The conversion of one molecule of glucose to two pyruvate in glycolysis generates two molecules of NADH. Since the amount of NAD.sup.+ in the cell is limited, it is essential for the continuation of glycolysis that NAD.sup.+ is rapidly reformed from the NADH produced. The inner mitochondrial membrane is impermeable to oxaloacetate, NADH and NAD.sup.+, and there is no mitochondrial transport system for these molecules. This means that mitochondrial oxaloacetate is not replenished from the cytosol, nor does the NADH formed in the cytosol by glycolysis have access to the electron transfer chain. Depletion of mitochondrial NAD.sup.+ alters the low NA.sup.+ /NADH ratio essential for the reduction of the electron transfer chain flavin carriers. The biochemical impasse is resolved by the cell through the use of `substrate shuttles` which transport hydrogen atoms across the mitochondrial membranes. These shuttles comprise a reaction between NADH and an oxidized substrate in the cytosol, followed by transport of the reduced substrate into the mitochondrion. The reduced substrate is then oxidized by the electron transfer chain.
Elimination of toxic hydrogen peroxide which is synthesized as a metabolic byproduct within mitochondria is modulated by the reductant glutathione. Oxidized glutathione produced in some of these processes is reduced by NADPH through the action of glutathione reductase. Since NADPH cannot be transported through the inner mitochondrial membrane, it must be regenerated from endogenous NADP.sup.+ to maintain low NADP.sup.+ /NADPH ratios. In erythrocytes, which have no mitochondria, NADPH is regenerated by glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase. Glutamate dehydrogenase may be responsible for the regeneration of NADPH in the mitochondria, but this has not yet been proven. Cytosolic malate dehydrogenase converts malate and NADP.sup.+ to pyruvate and NADPH as part of the pyruvate-malate shuttle. This shuttle is part of a pathway which enables mitochondrial acetyl coenzyme A to be transported via citrate to the cytosol to sites of fatty acid synthesis. A mitochondrial metabolic pathway in which malate is converted to pyruvate, with the concomitant conversion of NADP.sup.+ to NADPH serves to regenerate NADPH and to increase levels of mitochondrial pyruvate in the absence of pyruvate import from the cytosolic sources.
Malate dehydrogenase, an NAD(P).sup.+ -dependant dehydrogenase, in cooperation with aspartate aminotransferase isozymes, plays a pivotal role in the malate-aspartate shuttle and the pyruvate-malate shuttle. Regeneration of either mitochondrial NADH or NADPH is effected through the conversion of endogenous malate to pyruvate catalyzed by malate dehydrogenase. Four isoforms of the enzyme have been isolated from human tissue. Two human NAD.sup.+ -dependant malate dehydrogenase isoforms have been identified; one form is present in smooth muscle and striated muscle cytoplasm, the other in the mitochondria from rapidly proliferating and tumor cells (Tanaka, T. et al. (1996) Genomics 32:128-130; Loeber, G. et al. (1991) J. Biol. Chem. 266:3016-3021). Two NADP.sup.+ -dependant isoforms have also been identified in human breast cancer cell cytoplasm and in human hippocampal mitochondria (Chou, W. Y. (1996) J. Protein Chem. 15:272-279; Loeber, G. et al. (1994) Biochem. J. 304:687-692).
Most mitochondrial proteins, including malate dehydrogenase, are encoded by nuclear DNA. The enzyme is synthesized as a larger precursor molecule and subsequently transported into the mitochondria. An N-terminal region mediates recognition of protein targeted for this organanelle and is termed the "transit peptide". Upon binding and import to the mitochondrion, the transit peptide is removed by proteolysis and the subunits assemble to form active complexes (Grant, P. M. et al. (1986) Nucleic Acids Res. 14:6053-6066).
Two genes encoding murine malate dehydrogenase isoforms have been identified; one is a cytosolic isoform from heart and liver and the other is a mitochondrial isoform from liver. The protein products share 23% homology. Levels of mRNA encoding the mitochondrial isoform are elevated in heart, brain, and kidney, and are relatively low in liver (Joh, T. et al. (1987) Biochemistry 26:2515-2520; Joh, T. et al. (1987) J. Biol. Chem. 262:15127-15131).
The binding sites for NAD.sup.+ and NADP.sup.+ are similar in dehydrogenase enzymes isolated from organisms as diverse as bacteria, yeast, insects, and mammals, but those for NADP.sup.+ differ from those that bind NAD.sup.+ at certain amino acid residue positions. These differences are to accommodate the different charge distributions within the nicotinamide adenine dinucleotide substrates (Scrutton, N. S. et al. (1990) Nature 343:38-43).
Reduced mitochondrial malate dehydrogenase activity in polymorphonuclear cells has been associated with 7-monosomy myelodysplastic syndrome, and in peripheral blood leukocytes (PBL) from Duchenne muscular dystrophy (Muchi, H. and Yamamoto, Y (1983) Blood 62:808-814; Wisniewska, W. and Lukasiuk, M. (1985) Neurol. Neurochir. Pol. 19:318-322). Significantly increased levels of mitochondrial malate dehydrogenase have been found in human breast cancer tissue, in PBL following myocardial infarction, and in PBL associated with hepatocarcinoma and acute circulatory failure (Balinsky, D. et al. (1984) J. Natl. Cancer Inst. 72:217-224; Wagenknecht, K. et al. (1988) Kardiologiia 28:55-57; Kawai, M. and Hosaki, S. (1990) Clin. Biochem. 23:327-334).
The discovery of a new human mitochondrial malate dehydrogenase and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention and treatment of developmental, vesicle trafficking, immunological, and neoplastic disorders.